Research Summary

Research in the Riethman laboratory focuses on analyzing the structure, function, and evolution of mammalian telomere regions.

Introduction

Telomeres are dynamic and complex chromosomal structures. They are essential for genome stability and faithful chromosome replication, and mediate key biological activities including cell cycle regulation, cellular aging and immortalization, movements and localization of chromosomes within the nucleus, and transcriptional regulation of subtelomeric genes. The DNA at each human chromosome terminus is a simple repeat sequence tract (TTAGGG)n, typically 5 kb to 15 kb in length in somatic cells, that ends with a single-stranded extension of the G-strand of DNA The lengths of the terminal repeat tracts are dynamically modulated in a tissue-specific and individual-specific manner; loss of this sequence tract is correlated with cellular and organismal aging, and continuous maintenance of this tract is essential for cellular immortalization and cancer progression. Adjacent to this “terminal repeat” is a subtelomeric repeat region comprised of a mosaic patchwork of segmentally duplicated DNA. This class of low-copy repeat DNA is characterized by very high sequence similarity (90 % to >99.5 %) between duplicated tracts, and variably-sized but often very large duplicated segment lengths (1 kb to > 200 kb). The aggregate size of a subtelomeric repeat region varies according to the specific telomere; the shortest subtelomeric repeat region is 2 kb in length and the longest is greater than 500 kb. At many individual telomeres, allelic differences in the sizes of subtelomeric repeat regions can be large, sometimes on the order of hundreds of kilobases in length.

The large copy number polymorphisms (CNPs) and unusual sequence organization has complicated the cloning, mapping, and sequencing of human subtelomeres. These same properties make subtelomere-associated regions especially prone to deletions, translocations, and other DNA rearrangements, and has led to their rapid evolution. For example, evolutionarily recent duplicative translocations of large subtelomeric DNA tracts has led to the generation of new gene families in primates, and to the formation of novel fusion transcripts with potentially new functions. In addition, germline instability of subtelomeric DNA leads to human diseases; the deletion of a segment of subtelomeric repeat DNA near the 4q telomere is closely linked with the genetic disease FSHD, terminal deletions of 16p result in alpha-thalassemia, and an estimated 5-10% of all cases of idiopathic mental retardation are associated with cryptic rearrangements of subtelomeric DNA. An important unanswered question is whether/how the large subtelomeric structural variations contribute to genome instability and to both normal and disease phenotypes.

Proper maintenance of the terminal (TTAGGG)n DNA tract is critical for cell division and genome stability. Telomere shortening and/or dysfunction triggers cellular senescence or apoptosis in cells with intact checkpoint pathways. However, if these checkpoints are bypassed, continued telomere shortening leads to genome instability, crisis, and reactivation of telomere maintenance pathways which permit cellular immortalization and cancer. Human aging correlates both with shortened telomeres and with a dramatic rise in cancer. More generally, individuals with telomeres shorter than age-matched controls are susceptible to a range of diseases and have a higher mortality. (TTAGGG)n tract lengths are telomere-specific and regulated in part by cis-acting subtelomeric DNA. Our lab is developing and refining methods for accurately tracking individual subtelomeric alleles in human populations, and for measuring (TTAGGG)n tract lengths at individual human telomeres. This capability may reveal inborn individual-specific differences in telomere lengths that could affect susceptibility to aging and cancer, and would facilitate investigation of human telomere function and dysfunction in molecular detail. A key scientific question for us is whether/how cellular senescence relates to organismal aging and age-related pathologies including cancer and heart disease.

Riethman Lab Contributions

Cloning, mapping, and collaborative DNA sequencing efforts culminated in reference sequences for each of the 41 genetically distinct human subtelomeric regions (Riethman et al. 2001, 2004). Sequence gaps that remained on the reference telomeres were generally small, well-defined, and for the most part restricted to regions directly adjacent to the terminal (TTAGGG)n tract. Distal subtelomere regions are highly enriched in recently-duplicated chromosome segments relative the rest of the human genome. Our analysis of the substructure of this duplicated DNA has shown that (1) some duplicon blocks comprising the mosaic patchwork are very highly similar (98 to 99.5% identical), whereas others are less so; (2) Internal islands of (TTAGGG)n-like sequences, which are involved in controlling DNA replication and in enhanced recombination in model organisms, are enriched > 25-fold in subtelomeric DNA relative to the rest of the genome and are almost always located at duplicon boundaries; (3) While most duplicon blocks have copies in both internal and in subtelomeric regions, a subset localizes exclusively to subtelomeres – these subtelomere-specific blocks may provide an opportunity to develop probes for tracking particular subtelomeric CNPs; (4) subterminal duplicon blocks, which are immediately adjacent to terminal (TTAGGG)n tracts and are potentially involved in cis-regulation of allele-specific (TTAGGG)n tract length, group into just 6 families of sequences, the detailed characterization of which may permit development of subterminal genotyping assays.

Transcripts annotated in our subtelomere assemblies frequently span 1-copy/duplicon and duplicon/duplicon boundaries, indicating that the ongoing evolutionary shuffling of human subtelomeric genomic DNA segments is providing opportunities for the duplication and generation of new genes. The overall transcript density in subtelomere regions is similar (within about 10%) to that which is found genome-wide, but there is wide variability in gene density among individual telomeres. Zinc finger-containing genes, olfactory receptor genes, and many additional transcript families of unknown function were found to be duplicated within and between multiple human telomere regions. Comparative genomic approaches with non-human primate subtelomeres could reveal how human subtelomeres evolved and point towards functionally important subtelomeric sequence elements.

Telomeric and subtelomeric DNA regions exhibit a high level of structural variation that is likely to impact telomere function but has yet to be characterized in detail. A resource of clone libraries and associated paired-end reads constructed as part of the Human Structural Variation initiative is being used along with a combination of computational and wet-lab mapping methods to identify and characterize structural variants in distal human subtelomere regions. Terminal fosmids from these libraries were identified, mapped and used to characterize and sequence (TTAGGG)n-adjacent subterminal DNA. Identification and characterization of telomeric structural variants will close many of the remaining gaps in the human genome sequence, and will in the long term reveal the universe of common germline subterminal allele structures that exist. The global complement of variant subtelomeric alleles in a given individual may have important consequences for expression in gene-rich subtelomeric regions, and could contribute substantially to both natural human phenotypic variation and to disease phenotypes. Appropriately regulated (TTAGGG)n tracts are critical for normal cell function; individual (TTAGGG)n tract lengths in humans are allele-specific and regulated in part by cis-acting subtelomeric elements. The sequence information on subterminal alleles and allelic variants will open new avenues of telomere research and provide novel opportunities for developing methods to track subterminal genotypes in populations and measure allele-specific telomere lengths.

Current Work

The Riethman laboratory is focused on the detailed characterization of genetic and epigenetic features of human subtelomeric and telomeric DNA as they relate to cancer and aging. This work is currently in two main areas. In the first, novel experimental and computational next-gen sequencing based approaches are being developed and used for analysis of subtelomeric structural variation, mutation, and annotation of human subtelomeric DNA. The Riethman laboratory, in collaboration with the Lieberman and Davuluri labs at Wistar, has used existing next-gen datasets to annotate our updated human subtelomere sequence assemblies as part of an effort to understand the role of subtelomeric chromatin in the transcriptional regulation and function of a long noncoding RNA (TERRA) essential for telomere integrity (Deng et al., 2012). We have recently extended and refined our computational pipeline to enable efficient mapping of next-gen datasets to extended subtelomere DNA regions, where critically important genes are located and where previous analyses have been hampered by subtelomeric segmental duplication content. In the second area, the relationship of telomere length dynamics with telomere function and dysfunction is being investigated in several distinct contexts. Our laboratory is measuring telomere length as an environmentally-impacted biomarker associated with prostate cancer incidence and progression (collaboratively with Dr. T. Rebbeck and colleagues at University of Pennsylvania). In addition, we are developing both next-gen sequence-based and single-molecule fluorescence-based approaches to detect and characterize mutated and dysfunctional telomeres in cancer cells as well as in cells of individuals bearing constitutional ring chromosomes (collaboratively with Dr. Ming Xiao at Drexel and Dr. Nancy Spinner at CHOP). Finally, we are developing a novel method for the purification and nanomap-assisted sequencing of subtelomeric DNA from any genomic source, for the characterization of new subtelomeric structural variants (in collaboration with Dr. Ming Xiao, Drexel). Successful application of these methods will dramatically improve the quality and alternative allele depth of subtelomeric regions in the current human reference sequence, and open the door to telomeric DNA fragment capture and characterization from uncloned genomic DNA to permit population-based studies of the role of these sequences in telomere function.

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The microscope in the image belonged to William E. Horner, M.D., a collaborator with Caspar Wistar, M.D., in the early 1800s.

Dr. Horner, a lecturer at the University of Pennsylvania, was a pioneer of the use of microscopes in anatomical and medical research. He authored Special Anatomy and Histology, a seminal text on the subject.